Cytosine Demethylation Research Articles

Cytosine Methylation and Demethylation Model: Equilibrium Point and the Influence of Enzymes on Cytosine Forms Levels

Epigenetic changes, apart from changes in the DNA sequence, are one of the most important factors determining the fate of cells in living organisms. The main epigenetic change is cytosine methylation in relevant regions of DNA. Recently, we published an article in which we proposed a mathematical model of cytosine methylation and demethylation. In present work, we present an equilibrium analysis of this model. We present the conditions for its stability and aperiodic nature of the system’s free response. In particular, we focus on the influence of the levels of enzymes involved in cytosine methylation and demethylation on the equilibrium coordinates. The conclusions from this analysis are important in understanding the biological basis of the behavior of cells in living organisms.

Revealing the effects of two DNA methyltransferase inhibitors on DNA methylation and carotenoid metabolism in Chlamydomonas reinhardtii by comparative transcriptome and physiological analysis

DNA methylation plays an important role in cell growth and development. However, little is known about the possible functions of DNA methylation in microalgae. Here, two DNA methyltransferase (DNMT) inhibitors (5-azacytidine and zebularine) were used to treat Chlamydomonas reinhardtii to investigate the effects of DNA methylation on microalgae. 5-Azacytidine (5-Azac) showed the promoting effects on cell growth and was benefit to the accumulation of pigments, while zebularine (Zeb) has adverse effects on the physiology of algal cells. However, the genomes of C. reinhardtii treated with two DNMT inhibitors unexpectedly showed slightly increased 5-mC levels. According to the results of transcriptome sequencing to algal cells treated with Zeb and 5-Azac, two DNMT inhibitors were found to up-regulate the transcription levels of genes involved in the cytosine methylation pathway and down-regulate genes involved in the cytosine demethylation pathway, which may result in higher 5-mC levels in genome. The increased content of carotenoids by 5-Azac may be due to the up-regulated transcription levels of carotenogenic genes. In addition, the up-regulated transcription levels of carotenoid degradation-related genes may be responsible for the reduced carotenoid content by Zeb. Finally, a carotenoid 9,10(9′,10′)-cleavage dioxygenase 1 (CrCCD1) gene was isolated from C. reinhardtii, and its important role in carotenoid degradation was demonstrated. In summary, this study revealed the physiological changes and molecular mechanisms of C. reinhardtii under the effects of two DNMT inhibitors, providing valuable information for subsequent studies on DNA methylation in microalgae.

Advances in high throughput sequencing methods for DNA damage and repair.

With the rapid development of high-throughput sequencing technology in the past decade, an increasing number of sequencing methods targeting different types of DNA damage have been developed and widely used in the field. These technologies not only help to elucidate the dynamic processes of repair pathways corresponding to different types of lesions, understand the underlying mechanisms of key factors and identify new hotspots prone to damage, but also greatly advanced our knowledge of crucial physiological processes such as meiotic homologous recombination, antibody generation and cytosine demethylation. These advancements hold significant potential for broader applications in exploring disease initiation and drug development. However, understanding and selecting the appropriate techniques have become difficult. This article reviews the main sequencing detection methods for the most common DNA lesions and introduce their principles, thereby providing valuable insights for the selection, application, further development and optimization of these technologies.

Transcriptome variations in hybrids of wild emmer wheat (Triticum turgidum ssp. dicoccoides)

BackgroundWild emmer wheat is a great candidate to revitalize domesticated wheat genetic diversity. Recent years have seen intensive investigation into the evolution and domestication of wild emmer wheat, including whole-genome DNA and transcriptome sequencing. However, the impact of intraspecific hybridization on the transcriptome of wild emmer wheat has been poorly studied. In this study, we assessed changes in methylation patterns and transcriptomic variations in two accessions of wild emmer wheat collected from two marginal populations, Mt. Hermon and Mt. Amasa, and in their stable F4 hybrid.ResultsMethylation-Sensitive Amplified Polymorphism (MSAP) detected significant cytosine demethylation in F4 hybrids vs. parental lines, suggesting potential transcriptome variation. After a detailed analysis, we examined nine RNA-Seq samples, which included three biological replicates from the F4 hybrid and its parental lines. RNA-Seq databases contained approximately 200 million reads, with each library consisting of 15 to 25 million reads. There are a total of 62,490 well-annotated genes in these databases, with 6,602 genes showing differential expression between F4 hybrid and parental lines Mt. Hermon and Mt. Amasa. The differentially expressed genes were classified into four main categories based on their expression patterns. Gene ontology (GO) analysis revealed that differentially expressed genes are associated with DNA/RNA metabolism, photosynthesis, stress response, phosphorylation and developmental processes.ConclusionThis study highlights the significant transcriptomic changes resulting from intraspecific hybridization within natural plant populations, which might aid the nascent hybrid in adapting to various environmental conditions.

IPS cell generation-associated point mutations include many C > T substitutions via different cytosine modification mechanisms

Genomic aberrations are a critical impediment for the safe medical use of iPSCs and their origin and developmental mechanisms remain unknown. Here we find through WGS analysis of human and mouse iPSC lines that genomic mutations are de novo events and that, in addition to unmodified cytosine base prone to deamination, the DNA methylation sequence CpG represents a significant mutation-prone site. CGI and TSS regions show increased mutations in iPSCs and elevated mutations are observed in retrotransposons, especially in the AluY subfamily. Furthermore, increased cytosine to thymine mutations are observed in differentially methylated regions. These results indicate that in addition to deamination of cytosine, demethylation of methylated cytosine, which plays a central role in genome reprogramming, may act mutagenically during iPSC generation.

The DNA Methyltransferase Inhibitor 5-Aza-4'-thio-2'-Deoxycytidine Induces C>G Transversions and Acute Lymphoid Leukemia Development.

DNA methyltransferase inhibitors (DNMTi), most commonly cytidine analogs, are compounds that decrease 5'-cytosine methylation. DNMTi are used clinically based on the hypothesis that cytosine demethylation will lead to re-expression of tumor suppressor genes. 5-Aza-4'-thio-2'-deoxycytidine (Aza-TdCyd or ATC) is a recently described thiol-substituted DNMTi that has been shown to have anti-tumor activity in solid tumor models. In this study, we investigated the therapeutic potential of ATC in a murine transplantation model of myelodysplastic syndrome. ATC treatment led to the transformation of transplanted wild-type bone marrow nucleated cells into lymphoid leukemia, and healthy mice treated with ATC also developed lymphoid leukemia. Whole-exome sequencing revealed 1,000 acquired mutations, almost all of which were C>G transversions in a specific 5'-NCG-3' context. These mutations involved dozens of genes involved in human lymphoid leukemia, such as Notch1, Pten, Pax5, Trp53, and Nf1. Human cells treated in vitro with ATC showed 1,000 acquired C>G transversions in a similar context. Deletion of Dck, the rate-limiting enzyme for the cytidine salvage pathway, eliminated C>G transversions. Taken together, these findings demonstrate a highly penetrant mutagenic and leukemogenic phenotype associated with ATC. Significance: Treatment with a DNA methyltransferase inhibitor generates a distinct mutation signature and triggers leukemic transformation, which has important implications for the research and clinical applications of these inhibitors.

The Role of Different TET Proteins in Cytosine Demethylation Revealed by Mathematical Modeling.

In living cells, some reactions can be conducted by more than one enzyme and sometimes it is difficult to establish which enzyme is responsible. Such is the case with proteins from the TET family, capable of converting 5-methyl-2'-deoxycytidine (5-mdC) in DNA to 5-(hydroxymethyl)-2'-deoxycytidine (5-hmdC) and further to 5-formyl-2'-deoxycytidine (5-fdC) and 5-carboxy-2'-deoxycytidine (5-cadC). The estimation of the efficiency of particular TETs in particular oxidative reactions and different cell types is important but experimentally difficult. Here, we propose an approach with mathematical modeling in which methylation and known deoxycytidine modification pathways are presented by 343 possible model versions with assumed different combinations of TET1, 2, and 3 activities in different pathways. Model parameters were calculated on the basis of 5-mdC, 5-hmdC, 5-fdC, 5-cadC, and 5-hmdU levels experimentally assessed in five human cultured cell lines and previously published. Selection of the model versions that give in simulations the best average fit to experimental data suggested that not all TET proteins participate in all modification reactions and that TET3 activity may be especially important in the reaction of 5-fdC removal.

Ferritin heavy chain supports stability and function of the regulatory T cell lineage

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten–eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

The role of DNA methylation in human pancreatic neuroendocrine tumours.

Pancreatic neuroendocrine tumours (PNETs) are the second most common pancreatic tumour. However, relatively little is known about their tumourigenic drivers, other than mutations involving the multiple endocrine neoplasia 1 (MEN1), ATRX chromatin remodeler, and death domain-associated protein genes, which are found in ~40% of sporadic PNETs. PNETs have a low mutational burden, thereby suggesting that other factors likely contribute to their development, including epigenetic regulators. One such epigenetic process, DNA methylation, silences gene transcription via 5'methylcytosine (5mC), and this is usually facilitated by DNA methyltransferase enzymes at CpG-rich areas around gene promoters. However, 5'hydroxymethylcytosine, which is the first epigenetic mark during cytosine demethylation, and opposes the function of 5mC, is associated with gene transcription, although the significance of this remains unknown, as it is indistinguishable from 5mC when conventional bisulfite conversion techniques are solely used. Advances in array-based technologies have facilitated the investigation of PNET methylomes and enabled PNETs to be clustered by methylome signatures, which has assisted in prognosis and discovery of new aberrantly regulated genes contributing to tumourigenesis. This review will discuss the biology of DNA methylation, its role in PNET development, and impact on prognostication and discovery of epigenome-targeted therapies.

Global DNA 5-hydroxymethylcytosine level and its chromosomal distribution in four rye species.

The rye genome has a large size with a high level of cytosine methylation, which makes it particularly convenient for studying the occurrence of potential cytosine demethylation intermediates. Levels of global 5-hydroxymethylcytosine (5hmC) were analysed by enzyme-linked immunosorbent assay (ELISA) and mass spectrometry in four rye species: Secale cereale, Secale strictum, Secale sylvestre, and Secale vavilovii. The amount of 5hmC showed interspecific variation, and was also variable among organs, i.e. coleoptiles, roots, leaves, stems, and caryopses. 5-Formylcytosine (5fC), 5-carboxycytosine (5caC), and 5-hydroxymethyluracil (5hmU) were also found to be present in the DNA of all species; their global level varied among species and organs. The 5hmC level clearly correlated with the 5-methylcytosine (5mC) quantity. The mass spectrometry analysis carried out on the 5mC enriched fraction supported this relationship. Highly methylated sequences also contained higher amounts of 5fC and most of all 5hmU, but not 5caC. The analysis of the distribution of 5hmC in chromosomes distinctly indicated the co-localization of 5mC with 5hmC in the same chromosomal regions. The regularities in the levels of 5hmC and other rare modifications of bases in the DNA may indicate that they play a role in the regulation of the rye genome.

A new demethylase gene, OsDML4, is involved in high temperature-increased grain chalkiness in rice.

High temperature (HT) can affect the accumulation of seed storage materials and cause adverse effects on the yield and quality of rice. DNA methylation plays an important role in plant growth and development. Here, we identified a new demethylase gene OsDML4 and discovered its function in cytosine demethylation to affect endosperm formation. Loss of function of OsDML4 induced chalky endosperm only under HT and dramatically reduced the transcription and accumulation of glutelins and 16 kDa prolamin. The expression of two transcription factor genes RISBZ1 and RPBF was significantly decreased in the osdml4 mutants, which caused adverse effects on the formation of protein bodies (PBs) with greatly decreased PB-II number, and incomplete and abnormally shaped PB-IIs. Whole-genome bisulfite sequencing analysis of seeds at 15 d after pollination revealed much higher global methylation levels of CG, CHG, and CHH contexts in the osdml4 mutants compared with the wild type. Moreover, the RISBZ1 promoter was hypermethylated but the RPBF promoter was almost unchanged under HT. No significant difference was detected between the wild type and osdml4 mutants under normal temperature. Our study demonstrated a novel OsDML4-mediated DNA methylation involved in the formation of chalky endosperm only under HT and provided a new perspective in regulating endosperm development and the accumulation of seed storage proteins in rice.

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos.

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

TET enzymes regulate skeletal development through increasing chromatin accessibility of RUNX2 target genes

The Ten-eleven translocation (TET) family of dioxygenases mediate cytosine demethylation by catalyzing the oxidation of 5-methylcytosine (5mC). TET-mediated DNA demethylation controls the proper differentiation of embryonic stem cells and TET members display functional redundancy during early gastrulation. However, it is unclear if TET proteins have functional significance in mammalian skeletal development. Here, we report that Tet genes deficiency in mesoderm mesenchymal stem cells results in severe defects of bone development. The existence of any single Tet gene allele can support early bone formation, suggesting a functional redundancy of TET proteins. Integrative analyses of RNA-seq, Whole Genome Bisulfite Sequencing (WGBS), 5hmC-Seal and Assay for Transposase-Accessible Chromatin (ATAC-seq) demonstrate that TET-mediated demethylation increases the chromatin accessibility of target genes by RUNX2 and facilities RUNX2-regulated transcription. In addition, TET proteins interact with RUNX2 through their catalytic domain to regulate cytosine methylation around RUNX2 binding region. The catalytic domain is indispensable for TET enzymes to regulate RUNX2 transcription activity on its target genes and to regulate bone development. These results demonstrate that TET enzymes function to regulate RUNX2 activity and maintain skeletal homeostasis.

Bifunctional Role of Fe(II)/2OG-Dependent TET Family 5-Methylcytosine Dioxygenases and ALKBH2,3 in Modified Cytosine Demethylation

Three forms of methylated cytosines are present in the eukaryotic genome: 3-methylcytosine, 4-methylcytosine and 5-methylcytosine. 3-methylcytosines create methyl lesions, which impair local DNA function and flexibility, resulting in replication and transcription error. On the other hand, 5-methylcytosine is usually present at the gene promoter which blocks transcription and translation. Fe(II)/2OG-dependent nucleic acid-modifying enzymes are the class of enzymes responsible for the demethylation of these modified cytosines. ALKBH2 and 3 remove 3-methylcytosine via a one-step direct demethylation process. On the other hand, active demethylation of 5mC is initiated by Ten-Eleven Translocation (TET)-family dioxygenases. Via oxidative demethylation, TET1-3 converts 5mC into 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. Remarkably, recent findings demonstrate that ALKBH2,3 possess oxidative demethylation properties, along with direct demethylation. On the other hand, the TET family of enzymes possess direct demethylation properties along with oxidative demethylation. Here we review the importance of methylated cytosines in human DNA, their origin, function and removal. In addition, we discuss the recent findings of extraordinary flexibility of Fe(II)/2OG-dependent nucleic acid-modifying enzymes ALKBH2,3 and TET family of enzymes in cytosine demethylation, as well as their impact on epigenetics.

A Bayesian hierarchical model for improving measurement of 5mC and 5hmC levels: Toward revealing associations between phenotypes and methylation states.

5-hydroxymethylcytosine (5hmC) is a methylation state linked with gene regulation, commonly found in cells of the central nervous system. 5hmC is associated with demethylation of cytosines from 5-methylcytosine (5mC) to the unmethylated state. The presence of 5hmC can be inferred by a paired experiment involving bisulfite and oxidation-bisulfite treatments on the same sample, followed by a methylation assay using a platform such as the Illumina Infinium MethylationEPIC BeadChip (EPIC). Existing methods for analysis of the resulting EPIC data are not ideal. Most approaches ignore the correlation between the two experiments and any imprecision associated with DNA damage from the additional treatment. Estimates of 5mC/5hmC levels free from these limitations are desirable to reveal associations between methylation states and phenotypes. We propose a hierarchical Bayesian method called Constrained HYdroxy Methylation Estimation (CHYME) to simultaneously estimate 5mC/5hmC signals as well as any associations between these signals and covariates or phenotypes, while accounting for the potential impact of DNA damage and dependencies induced by the experimental design. Simulations show that CHYME has valid type 1 error and better power than a range of alternative methods, including the popular OxyBS method and linear models on transformed proportions. Other methods we examined suffer from hugely inflated type 1 error for inference on 5hmC proportions. We use CHYME to explore genome-wide associations between 5mC/5hmC levels and cause of death in postmortem prefrontal cortex brain tissue samples. These analyses indicate that CHYME is a useful tool to reveal phenotypic associations with 5mC/5hmC levels.

TET2-mediated epigenetic reprogramming of breast cancer cells impairs lysosome biogenesis.

Methylation and demethylation of cytosines in DNA are believed to act as keystones of cell-specific gene expression by controlling the chromatin structure and accessibility to transcription factors. Cancer cells have their own transcriptional programs, and we sought to alter such a cancer-specific program by enforcing expression of the catalytic domain (CD) of the methylcytosine dioxygenase TET2 in breast cancer cells. The TET2 CD decreased the tumorigenic potential of cancer cells through both activation and repression of a repertoire of genes that, interestingly, differed in part from the one observed upon treatment with the hypomethylating agent decitabine. In addition to promoting the establishment of an antiviral state, TET2 activated 5mC turnover at thousands of MYC-binding motifs and down-regulated a panel of known MYC-repressed genes involved in lysosome biogenesis and function. Thus, an extensive cross-talk between TET2 and the oncogenic transcription factor MYC establishes a lysosomal storage disease-like state that contributes to an exacerbated sensitivity to autophagy inducers.

The N-terminal domain of TET1 promotes the formation of dense chromatin regions refractory to transcription.

TET (ten-eleven translocation) enzymes initiate active cytosine demethylation via the oxidation of 5-methylcytosine. TET1 is composed of a C-terminal domain, which bears the catalytic activity of the enzyme, and a N-terminal region that is less well characterized except for the CXXC domain responsible for the targeting to CpG islands. While cytosine demethylation induced by TET1 promotes transcription, this protein also interacts with chromatin-regulating factors that rather silence this process, the coordination between these two opposite functions of TET1 being unclear. In the present work, we uncover a new function of the N-terminal part of the TET1 protein in the regulation of the chromatin architecture. This domain of the protein promotes the establishment of a compact chromatin architecture displaying reduced exchange rate of core histones and partial dissociation of the histone linker. This chromatin reorganization process, which does not rely on the CXXC domain, is associated with a global shutdown of transcription and an increase in heterochromatin-associated histone epigenetic marks. Based on these findings, we propose that the dense chromatin organization generated by the N-terminal domain of TET1 could contribute to restraining the transcription enhancement induced by the DNA demethylation activity of this enzyme.

Prognostic Utility of CpG Island Hypermethylated Phenotype in Early-Stage Invasive Primary Melanomas

In their article in the Journal of Investigative Dermatology, Conway etal. (2021) show the significance of the CpG island methylator phenotype (CIMP) subclass in early-stage invasive primary melanomas, particularly in the lentigo maligna melanoma histological subtype. CIMP melanomas had reduced melanoma-specific survival compared with intermediate and low methylation subtypes. This study emphasizes the importance of epigenomic changes in early-stage primary tumors that are associated with poor prognosis.

DNA Methylation and Establishing Memory.

A single event can cause a life-long memory. Memories physically reside in neurons, and changes in neuronal gene expression are considered to be central to memory. Early models proposed that specific DNA methylations of cytosines in neuronal DNA encode memories in a stable biochemical form. This review describes recent research that elucidates the molecular mechanisms used by the mammalian brain to form DNA methylcytosine encoded memories. For example, neuron activation initiates cytosine demethylation by stimulating DNA topoisomerase II beta (TOP2B) protein to make a temporary DNA double-strand break (repaired within about 2 hours) at a promoter of an immediate early gene, EGR1, allowing expression of this gene. The EGR1 proteins then recruit methylcytosine dioxygenase TET1 proteins to initiate demethylation at several hundred genes, facilitating expression of those genes. Initiation of demethylation of cytosine also occurs when OGG1 localizes at oxidized guanine in a methylated CpG site and recruits TET1 for initiation of demethylation at that site. DNMT3A2 is another immediate early gene upregulated by synaptic activity. DNMT3A2 protein catalyzes de novo DNA methylations. These several mechanisms convert external experiences into DNA methylations and initiated demethylations of neuronal DNA cytosines, causing changes in gene expression that are the basis of long-term memories.

Abstract 2124: Vitamin C enhances H3K36me3 via NSD1 promoter demethylation and sensitizes GBM stem cells to DNA damaging agents

Abstract Primary brain tumors are among the most devastating forms of cancer. Due to profound genetic, epigenetic, and morphologic heterogeneity, GBM represents the most aggressive and lethal form of the disease. GBM exhibits aberrant patterns of DNA methylation, resulting in a highly distorted epigenome, yet the molecular circuits responsible for this epigenetic dysregulation remain unknown. Glioma stem cells (GSCs) are critical modulators of tumor propagation and therapeutic resistance in GBM, and epigenetic deregulation within this cell subpopulation is thought to play fundamental roles in GSC and GBM biology. Understanding and overcoming these tumorigenic epigenetic mechanisms in GSCs is crucial to developing more effective treatment protocols and improving clinical survival. DNA methylation is a reversible process involving the addition of methyl groups, typically, at cytosine residues and demethylation is catalyzed, in part, by the ten-eleven translocation (TET) family of enzymes. These enzymes function as deoxygenases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Multiple studies show negative correlations between 5hmC levels and glioma grade, and loss of 5hmC correlates with poor prognosis of GBM patients. These observations suggest that understanding the mechanisms of 5hmC deregulation in GBM and developing innovative ways to re-establish 5hmC levels in tumors can impact patient outcome. Ascorbate (Vitamin C) is a TET enzyme cofactor that has gained pre-clinical interest as an attractive antitumor agent for GBM due to its ability to regulate redox status, function as potent demethylating agent, and efficiently penetrate the blood-brain barrier. We show through various molecular and biochemical endpoints, that low-dose Vitamin C significantly replenishes 5hmC and decreases stemness in GSCs. We found that, in addition to increasing global levels of 5hmC, low-dose Vitamin C acts synergistically with temozolomide (TMZ) and irradiation to induce cell death in GSCs. We also present a novel mechanism whereby Vitamin C regulates promoter demethylation of the histone methyltransferase NSD1 resulting in an increase in H3K36me3 in GSCs. We propose the increase in H3K36me3 results in euchromatin states that are more susceptible to DNA damage therapies. Our findings strongly indicate that Vitamin C can undo tumor-promoting DNA hypermethylation and sensitize GSCs to conventional radio- and chemotherapeutics and suggest it might serve as an adjunct to standard-of-care GBM treatment. Citation Format: Harmon Khela, Sweta Sudhir, Maria Fagundo-Lugo, Bachchu Lal, John Laterra, Hernando Lopez-Bertoni. Vitamin C enhances H3K36me3 via NSD1 promoter demethylation and sensitizes GBM stem cells to DNA damaging agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2124.